Compositions, methods and products comprising human papillomavirus for detecting and treating a cancer

ABSTRACT

Methods for screening a patient for a cancer wherein the methods comprise detecting an HPV in a biopsy from a patient are disclosed. Also disclosed are compositions and products for screening and for treating cancer in a patient, as well as methods of treating a patient afflicted with a cancer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to products, compositions, methods andapparatus for identification of cancers and pre-cancerous cellularchanges. In another aspect, the present invention relates to products,compositions, methods and apparatus for identification of breast cancersor pre-cancerous cellular changes in breast tissues. In even anotheraspect, the present invention relates to products, compositions, methodsand apparatus for treatment of cancers and pre-cancerous cellularchanges. In still another aspect, the present invention relates toproducts, compositions, methods and apparatus for treatment of breastcancer and pre-cancerous cellular changes in breast tissues.

2. Description of the Related Art

Breast cancer is the most common form of cancer in women in the UnitedStates. It is estimated that in the year 2000, 182,800 new cases offemale invasive breast cancer will be diagnosed, and 40,800 women willdie from the disease. All women are at risk for breast cancer, with thisrisk increasing as a woman ages. Women are generally considered to be atincreased risk for developing breast cancer if they have one or more ofthe following risk factors: a) a family history of breast cancer, b) aprevious diagnosis of a malignant breast tumor or other gynecologicalcancers, c) hormonal factors, or d) not having had any children orhaving the first child later in their child bearing years. Even so, themajority of all breast cancers occur in women who apparently do not haveidentifiable risk factors.

Breast cancer cannot currently be prevented. But detecting and treatingit at an early stage, when the tumor is small and has not spread beyondthe breast, can increase the chances of survival significantly. However,not all breast cancers are currently detected at this early stage.Therefore, screening for breast cancer has become a critical aspect inthe overall management of this disease.

The techniques currently used to screen for breast cancer and otherbreast conditions include monthly breast self examination, mammography,and clinical breast examination. Also, genetic testing can be performedfor BRCA1 and BRCA2 genes in women who have a strong family history ofbreast cancer, since these genes are associated with approximately 5 to10 percent of breast cancer cases. In spite of this genetic knowledge,the genetic changes involved in the vast majority of breast cancersremains largely undetermined.

Human papillomaviruses (HPV) are strongly linked to cervical and othercancers. Cervical cancer (CX CA) is the second most prevalent femalecancer world-wide. HPV 16 DNA is present in 65% of CX CAs, and with theother HPV types, more than 90% of CX CAs contain HPV DNA. The E6 and E7genes of HPV 16s can cause contact-inhibited cells to lose thisphenotype. Furthermore, E6 and E7 interact with the cellularanti-oncogenes RB¹⁰⁵ and p53, respectively, leading to theirinactivation. Thus, it is widely regarded that HPV-16 is a centraletiologic agent and risk factor in the development of cervical/genitalcancer. The E7 protein, and possibly E6 as well, also function astranscriptional transactivators of heterologous genes. HPVs have alsobeen found in oral, penile, and vulvar cancer.

It appears that whatever tissue site HPVs are known to infect, theycause pathology. Usually the pathology is limited to a tissuehyperplasia or papilloma. However, there is a significant risk that thishigher than normal active cell growth may become an outright malignancy.

However, as recent studies have indicated, the relationship between HPVinfection and breast cancer is controversial.

Hennig et al. (1999) have reported that of women studied in Norwayhaving concomitant advanced genital HPV infection (cervicalintraepithelial neoplasia III, “CIN III”) in addition to breast cancer,46% of the breast cancers also contained HPV 16. However, of the controlstudy of eight patients having breast cancer diagnosed before the CINIII lesions, none had HPV positive breast carcinomas. Additionally nocases in the study were positive for HPV 11, 18 or 33.

Yu et al. (1999) report that HPV 33 is associated in pre-malignant andmalignant breast lesions in Chinese and Japanese populations, andfurther suggest that HPV 16 and HPV 18 are not involved in breasthyperplastic lesion, especially breast cancer.

In spite of the advancements in the art, there is a need in the art forimproved compositions, methods and products for screening a patient forcancer and/or pre-cancerous cellular changes.

There is another need in the art for improved compositions, products andmethods for screening a patient for breast cancer and/or pre-cancerouscellular changes in the breast.

There is even another need in the art for improved compositions,products and methods for treating a patient afflicted with a cancer inany stage of development.

There is still another need in the art for improved compositions,products and methods for treating a patient afflicted with breast cancerand/or a pre-cancerous cellular changes in the breast in any stage ofdevelopment.

These and other needs in the art will become apparent to those of skillin the art upon review of this specification, including its drawings,claims and appendix.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide compositions,methods and products for screening a patient for cancer and/orpre-cancerous cellular changes.

It is another object of the present invention to provide forcompositions, products and methods for screening a patient for breastcancer and pre-cancerous cellular changes in the breast.

It is even another object of the present invention to providecompositions, products and methods for treating a patient afflicted witha cancer and/or pre-cancerous cellular changes in any stage ofdevelopment

It is still another object of the present invention to providecompositions, products and methods for treating a patient afflicted withbreast cancer and/or a pre-cancerous cellular changes in the breast inany stage of development.

These and other objects in the art will become apparent to those ofskill in the art upon review of this specification, including itsdrawings, claims and appendix.

According to one embodiment of the present invention there is provided amethod of screening a patient for a cancer. The method generallycomprises performing an amplification technique on a sample from abiopsy taken from a patient. The sample comprises nucleic acid, and theamplification technique is directed to specific amplification of aportion of a human papillomavirus (HPV) sequence contained therein. Themethod further includes probing for the presence of an HPV sequence inthe amplified sequence using an HPV specific probe.

According to another embodiment of the present invention there isprovided a method of screening a patient for a cancer. The methodgenerally comprises contacting cellular material together with an HPVspecific probe. The cellular material is generally extracted from asample, such as a biopsy, taken from a patient. The cellular materialmay be any purified or non-purified cellular material such as, forexample, deoxyribonucleic acid (DNA), ribonucleic acid (RNA),polypeptides, or a combination thereof. The cellular material may bepurified, either partially or wholly, using any of the methods wellknown in the art.

The probe used in the screening methods of the invention may be specificto any HPV selected from the group consisting of HPV18, HPV31, HPV 33,HPV35, HPV45, HPV58. In a preferred embodiment, the screening methodfurther comprises contacting the cellular material with a second HPVspecific probe, wherein the first and second HPV are different from oneanother and are selected from the group consisting of HPV18, HPV31, HPV33, HPV35, HPV45, HPV58. Alternatively, in another embodiment, thescreening method further comprises contacting the cellular material witha second HPV specific probe, wherein the first HPV specific probe isspecific to HPV 16 and the second HPV specific probe is specific to atleast one HPV selected from the group consisting of HPV18, HPV31, HPV33, HPV35, HPV45, HPV58.

According to even another embodiment of the present invention there isprovided a method of treating a patient. The method generally comprisesadministering a composition comprising an effective amount of anantisense HPV DNA sequence to a patient. Preferably, the antisense HPVDNA sequence is expressed from a viral expression vector, such as anadeno-associated vector. The HPV may be any member of the HPV family,such as, for example, HPV16, HPV18, HPV31, HPV35, HPV45, HPV58, and anycombination thereof.

According to still another embodiment of the present invention there isprovided a method of treating a patient. The treatment method generallycomprises administering an effective amount of a composition to apatient, wherein the composition comprises an agent that inhibitsexpression of at least one HPV gene.

According to yet another embodiment of the present invention there isprovided a method of treating a patient. The treatment method generallycomprises administering an effective amount of a composition to apatient, wherein the composition comprises an agent that specificallyinhibits an HPV protein. Examples of HPV proteins to target forinhibition include the HPV16 E6 protein and the HPV16 E7 protein.Inhibition of a protein can be by any of the methods known in the art,such as, targeting with an antibody, inhibition of post-translationmodification, inhibition of protein stability and half-life. A preferredagent for use in the treatment method of the present invention is anantibody specific for interaction with an epitope of an HPV protein,such as HPV16 E6 protein or HPV16 E7 protein.

According to even still another embodiment of the present inventionthere is provided a method of treating a patient. The method comprisestransfecting dendritic cells (DCs) into a patient, wherein the dendriticcells have been altered to stably produce an HPV antigen. Preferably, arecombinant retrovirus such as for example an adeno-associated virus(AAV) that has been genetically manipulated to comprise a portion of anHPV antigen-encoding gene is used to infect monocyte precursors whichare then induced to differentiate into DCs. Differentiation of monocytesin DCs may be accomplished by treating the monocytes with at least onecytokine.

According to even yet another embodiment of the present invention thereis provided for a kit useful for screening a patient for a cancer.Generally the kit comprises a probe that is specific for the detectionof an HPV family member. The HPV-specific probe may be a single-strandedoligonucleotide sequence, a double-stranded oligonucleotide sequence, apolypeptide, or any combination thereof. The HPV may be any HPV familymember including, HPV16, HPV18, HPV31, HPV35, HPV45, HPV58, and anycombination thereof. In a preferred embodiment the HPV is HPV16 orHPV18. The probe may be used on any sample derived from a patient.

These and other embodiments of the present invention will becomeapparent to those of skill in the art upon review of this specification,including its drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Polymerase Chain Reaction (PCR)/Dot Blot Hybridizationanalysis for HPV-16/18/31 “super” probe.

FIG. 2 provides results from a PCR/Dot blot analysis for HPVs using anL1 targeting primer set and probing with HPV-16 sequences.

FIG. 3 is a Polymerase Chain Reaction (PCR)/Dot Blot Hybridizationanalysis for HPVs in breast cancer specimens using an L1 targetingprimer set and probing with HPV-18 probe.

FIG. 4 is a Polymerase Chain Reaction (PCR)/Dot Blot Hybridizationanalysis for HPVs in breast cancer specimens using an L1 targetingprimer set and probing with HPV-31 probe.

FIG. 5 provides results from a PCR/Dot blot analysis for HPVs using anE6-E7 junction targeting primer set and probing with HPV-16 sequences.

FIG. 6 provides results from a PCR/Dot blot analysis for HPVs using anE6-E7 junction targeting primer set and probing with HPV-18 sequences.

FIG. 7 is a PCR/Dot Blot Hybridization analysis for contaminatingplasmids by using a primer set which targets the pBR322/ColE1 oriregion.

FIG. 8A shows a structural map of the AAV/NE6/NEO (a.k.a.d16-95/E6^(p5)/NEO^(SV40)) virus with the names of the components at thetop.

FIG. 8B shows the analysis of various 293/vector producer cell lines.

FIG. 8C shows a titering analysis of the AAV/E6/Neo virus stock used inthis study.

FIG. 8D shows a graphic description of the experimental protocol.

FIG. 9 provides E6 mRNA expression in infected DC.

FIG. 10 provides the efficiency of Mo/DC-pulsing analyzed byintracellular staining.

FIG. 11 shows the amount of chromosomal integration by AAV/E6/Neo in DC.

FIG. 12 shows the early appearance of priming rosettes duringAAV-mediated priming.

FIG. 13 shows the cytotoxic response resulting from AAV vector andDOTAP-protein lipofection after 7 days of priming.

FIG. 14 is a two-color flow cytometric characterization of primed cellpopulations.

FIG. 15 is the characterization of DC at day 7 under differentconditions.

FIG. 16 provides the characterization of CD80 in pulsed DC.

DETAILED DESCRIPTION OF THE INVENTION

According to one embodiment of the present invention there is provided amethod of screening a patient for a cancer. The method generallycomprises performing an amplification technique on a sample from abiopsy taken from a patient. The sample comprises cellular material,preferably nucleic acid, and the amplification technique is directed tospecific amplification of a portion of a marker for a cancer. In apreferred embodiment, the marker is a human papillomavirus (HPV)sequence. In a more preferred embodiment, the cancer is breast cancer inany stage of development. The method may further include probing for thepresence of an HPV sequence in the amplified sequence using an HPVspecific probe.

The methods of the invention may comprise amplification of at least oneHPV sequence selected from the group consisting of HPV18, HPV31, HPV 33,HPV35, HPV45, HPV58, or any combination comprising at least HPVsequences. Suitable combinations include: HPV16 and at least any one ofthe group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58; HPV18and at least any one of the group consisting of HPV16, HPV31, HPV 33,HPV35, HPV45, HPV58; and at least any two of the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.

With respect to probing for a specific HPV sequence, the methods of theinvention may comprise probing for at least one HPV sequence selectedfrom the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58,or any combination comprising at least HPV sequences. Suitablecombinations include: HPV16 and at least any one of the group consistingof HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58; HPV18 and at least any oneof the group consisting of HPV16, HPV31, HPV 33, HPV35, HPV45, HPV58;and at least any two of the group consisting of HPV 18, HPV31, HPV 33,HPV35, HPV45, HPV58.

The samples used in the present invention may be obtained from a biopsyfrom a cervical intraepithelial neoplasia III (CIN III) positive patientor a CIN III negative patient. Preferably the sample is obtained from abiopsy from a patient who is not afflicted with CIN III (a non-CIN IIIpatient). Thus the sample may first be tested/assayed for the presenceof any CIN III-marker known in the art.

The sample may be derived from the patient by any method known in theart, such as, for example, any well known method for obtaining a biopsy,including the recently reported technique of breast duct lavage (DooleyW. C. et al., Lancet, 2001, 357(9265):1335-6; Evron, E. et al.,Obstetrics & Gynecology, 2001, 97(4):S2, both of which are incorporatedherein by reference). Numerous methods for obtaining a sample via biopsyare known in the art and include for example bite, brush, cone,cytological, aspiration, endoscopic, excisional, exploratory,incisional, percutaneous, punch, and surface biopsy. Breast duct lavage,also referred to as ductal lavage and intraductal lavage, is arelatively non-invasive procedure and enables the retrieval of breastepithelial cells that line the ductal/lobular systems of all milk ducts.Whereas a needle is used in aspiration biopsy, the technique of ductallavage comprises use of a microcatheter which is inserted into the milkducts through the nipple surface orifices. Saline is then flushedthrough the ducts to wash out epithelial cells for collection andfurther evaluation.

Preparation of samples for amplification is well known in the art, andany such technique may be used herein. Amplification methods are wellknown in the art and include techniques such as, for example, polymerasechain reaction (PCR) amplification and reverse transcription PCR(RT-PCR), as well as others. The amplified products may be detected andanalyzed using any of the numerous techniques well known in the art.

The amplification technique used herein may be specific foramplification of a portion of at least one HPV sequence selected fromthe group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58. Theamplification technique used herein may be specific for amplification ofa portion of at least two HPV sequences selected from the groupconsisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58. Theamplification technique used herein may be specific for amplification ofa portion of HPV16 and at least one HPV sequence selected from the groupconsisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.

Another embodiment of the present invention provides for a method ofscreening a patient for a cancer. The method generally comprisescontacting cellular material together with an HPV specific probe. Thecellular material is generally extracted from a sample, such as abiopsy, taken from a patient. The cellular material may be any purifiedor non-purified cellular material such as, for example, deoxyribonucleicacid (DNA), ribonucleic acid (RNA), polypeptides, or a combinationthereof. The cellular material may be purified, either partially orwholly, using any of the purification methods well known in the art.

The HPV probes useful in the screening methods of the present inventionmay be any type of probe useful in detecting the presence of HPV. Theseprobe types include, but are not limited to, a single-stranded ordouble-stranded oligonucleotide sequence complementary to the plus orminus strand of an HPV DNA sequence, a single-stranded ordouble-stranded oligonucleotide sequence complementary to a portion ofan HPV mRNA sequence, and an antibody specific to an epitope of an HPVprotein. Suitable examples of HPV proteins include, but are not limitedto, the HPV16 E6 or HPV16 E7. Oligonucleotide sequences specific to HPVsequences are known in the art, such as, for example, those disclosed inBreast Cancer Rch. Trtmt, 53;121-135, Anticancer Rch. 19;5057-5062, J.Gen. Virol. 76:1057-1062, J. Pathol 165: 301-309, and J. Clin.Microbiol. 34: 2095-2100, with all of these articles herein incorporatedby reference. Oligonucleotides may also be designed according to the LosAlamos National Laboratory Database nomenclature for the different HPVgenomes, incorporated herein by reference.

Methods of detecting targeted sequences with a probe are well known inthe art and are included herein. Any type of hybridization method issuitable for use in the present invention.

The screening methods of the present invention may be performed on asample from any organism/patient capable of developing cancer.Preferably, the method of the present invention is performed on asamples taken from a mammal, more preferably a human. The patients onwhich the methods of the invention are used may be CIN III positive orCIN III negative. In a preferred embodiment, the patients are notafflicted with CIN III (CIN III negative). The screening methodsdescribed herein are useful in detecting numerous types of cancer, suchas, for example, breast, dermal, oral, penile, vulvar cancer, and anycombination thereof. In addition, the screening methods of the presentinvention are useful in detecting a cancer in any stage of development.

Even another embodiment of the present invention provides for a methodof treating a patient afflicted with a cancer. The method generallycomprises administering a composition comprising an effective amount ofan antisense HPV sequence to a patient. The size of the sequence is notlimited and can range in size from that of an oligonucleotide to that ofa transcript. The antisense HPV sequence may comprise DNA, RNA,ribosomal RNA, or any combination thereof. The HPV may be any member ofthe HPV family, non-limiting examples of which include, HPV16, HPV18,HPV31, HPV33, HPV35, HPV45, HPV58, and any combination thereof.Preferred HPV's include HPV 16 and HPV 18. Non-limiting examples ofcombinations include: HPV 16 with at least one of HPV18, HPV31, HPV33,HPV35, HPV45, and HPV58; HPV 18 with at least one of HPV16, HPV31,HPV33, HPV35, HPV45, and HPV58; both HPV16 and HPV18 with at least oneof HPV31, HPV33, HPV35, HPV45, and HPV58, and at least any two of HPV16,HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58.

The HPV sequence may be expressed from a recombinant expression vector.Suitable vectors are known in the art and include, for example,mammalian expression vectors and viral vectors. Examples of viralvectors suitable for use in the present invention include: retroviruses;adenoviruses; adenoviral/retroviral chimeras; adeno-associated viruses;herpes simplex virus I or II; parvovirus; and reticuloendotheliosisvirus. Other possible viral vectors may be derived from poliovirus,papillomavirus, vaccinia virus, lentivirus, as well as chimeric vectorsincorporating favorable aspects of any two or more of the above viruses.Preferably, the antisense HPV sequence is expressed from a recombinantviral expression vector, such as an adeno-associated vector.

Still another embodiment of the present invention provides for a methodof treating a patient afflicted with a cancer. The treatment methodgenerally comprises administering an effective amount of a compositionto a patient, wherein the composition comprises an agent that inhibitsexpression of at least one HPV gene. The patients of the presentinvention may be CIN III positive or CIN III negative. Preferably, thepatient is negative for CIN III.

Mechanisms for inhibiting the expression of a gene are numerous and wellknown in the art and include, but are not limited to, inhibiting genetranscription, inhibiting the messenger RNA (mRNA) of a gene, inhibitingtranslation of an mRNA, inhibiting post-translational modification of agene product, and inhibiting a gene product. These inhibition methodsmay be direct or indirect. Any of these mechanisms may be used in thepresent invention.

Agents that inhibit expression of at least one HPV gene suitable for usein the present invention include, an oligonucleotide or longer stretchof nucleic acid comprising antisense HPV DNA, RNA or ribosomal RNA, andan oligonucleotide or longer stretch of DNA, RNA, or ribosomal RNAcomprising a sequence complementary to the plus or the minus strand ofHPV DNA. The HPV targeted in the present invention may be any HPV familymember, non-limiting examples of which include, HPV16, HPV18, HPV31,HPV33, HPV35, HPV45, HPV58, and any combination thereof. Preferred HPV'sinclude HPV 16 and HPV 18. Non-limiting examples of combinationsinclude: HPV 16 with at least one of HPV18, HPV31, HPV33, HPV35, HPV45,and HPV58; HPV 18 with at least one of HPV16, HPV31, HPV33, HPV35,HPV45, and HPV58; both HPV16 and HPV18 with at least one of HPV31,HPV33, HPV35, HPV45, and HPV58, and at least any two of HPV16, HPV18,HPV31, HPV33, HPV35, HPV45, and HPV58.

Administration of the compositions of the present invention to arecipient may be by any method known in the art. Thus, administration ofthe present invention to a recipient may be by a route selected fromoral, parenteral (including, subcutaneous, intradermal, intramuscular,and intravenous) and rectal. For increased efficacy, the compositions ofthe present invention may be administered via localized delivery to thetargeted tissue, such as, for example, breast tissue in the case ofbreast cancer. A modified breast duct lavage technique, also known asductal lavage, may be used for localized directly of the anticancercompounds and compositions of the present invention to a breast duct andbreast epithelial cells. Similar to the technique for ductal lavage, amicrocatheter may be inserted into a nipple surface orifice andsubsequently into a milk duct. However, instead of simply flushing theduct in order to obtain epithelial cells, as is done with ductal lavage,the microcatheter may be used for localized delivery of a composition ofthe invention directly to the breast duct and breast epithelial cells.

Yet another embodiment of the present invention provides for a method oftreating a patient afflicted with a cancer. The cancer may be any cancerin any stage of development. In a preferred embodiment, the cancer isbreast cancer. In addition, the patient may be CIN III positive or CINIII negative. Preferably, the patient is negative for CIN III. Thetreatment method generally comprises administering an effective amountof a composition to a patient, wherein the composition comprises anagent that specifically inhibits an HPV protein. Examples of HPVproteins to target for inhibition include the HPV16 E6 protein and theHPV16 E7 protein. Inhibition of a protein can be by any of the methodsknown in the art, such as, inhibition of gene expression, targeting aprotein with an antibody, inhibition of post-translation modification,and inhibition of protein stability and half-life. A preferred agent foruse in the treatment method of the present invention is an antibodyspecific for interaction with an epitope of an HPV protein, such asHPV16 E6 protein or HPV16 E7 protein.

Additional inhibitory agents suitable for use in the compositions andmethods of the invention include agents wherein the agent is a DNA,cDNA, RNA, ribosomal RNA, or polypeptide sequence. Suitable examples ofsuch agents include, an antisense HPV sequence which inhibitstranscription or translation of a HPV gene or gene product,transcription factors which decrease expression of an HPV gene, factorswhich affect translation of an HPV mRNA, factors which decrease thestability/half-life of an HPV mRNA molecule, factors which decrease thestability/half-life of an HPV polypeptide, and factors which interactwith an HPV polypeptide, such as a polypeptide encoding an antibodywhich specifically interacts with an epitope of a HPV. The material andmethods for producing these types of inhibitors (DNA, cDNA, RNA andpolypeptide) are known in the art and are included in the presentinvention.

For example, expression vectors expressing a sequence inhibitory totranscription of a HPV gene or expressing a sequence inhibitory totranslation of a HPV mRNA are within the scope of the HPV inhibitorsdefined herein. Expression vectors suitable for the present inventionmay comprise an antisense HPV sequence, or a sequence encoding anegative regulator of transcription of a HPV gene.

Even still another embodiment of the present invention provides for amethod of treating a patient afflicted with a cancer. The methodgenerally comprises transfecting dendritic cells (DCs), primed T cellsor a combination thereof, into a patient, wherein the dendritic cellshave been altered to stably produce an HPV antigen. The basis for usingDC cells for human immunotherapy has recently been established asdescribed in Young, J. W., and Inaba, K. (1996) DCs as adjuvants forclass I manor histocompatibility complex-restricted antitumor immunity.J. Exp. Med. 183:7-11, and Santin, A. D., Hermonat, P. L., Ravaggi, Al,Chiriva-Internati, M., Hiserotdt, J. C., Pecorelli, S., and Parham, G.P. (1999) Kinetics of expression of surface antigens during thedifferentiation of human dendritic cells versus macrophages.Immunobiology 200, both of which are incorporated herein by reference.

Briefly, DCs are presently believed to be the most effective antigenpresenting cells for activating naive T cells. Blood monocytes can beinduced to differentiate in to DCs by treatment with at least onecytokine. DCs when pulsed with antigen (Ag) results in a class Irestricted cytotoxic response against the Ag. In most DC-pulsingprotocols of the art, antigen proteins to be targeted are transfectedinto DCs for immune stimulation. These DC protocols may be used herein.However, both the antigen to be presented by the Dcs, and the cytokinesused to generate the DCs degrade with time as a reflection of theirhalf-lives. Thus, a more effective technique for pulsing/treating theDCs may be the in situ generation/production of the antigen protein suchas at least a portion of an HPV protein, and/or a cytokine within and bythe DC itself. By delivering a tumor-antigen gene and/or cytokine genedirectly in the DC, a stable and continuous production of the proteinsmay be achieved.

Thus, a recombinant viral vector that has been genetically manipulatedto comprise a portion of a HPV gene and thus expresses at least aportion of an HPV gene may be used to infect monocyte precursors whichhave been acquired from a blood sample of a patient. The infectedmonocytes are then induced to differentiate into DCs by treatment withat least one cytokine. Examples of suitable cytokines include, but arenot limited to IL-2, IL-4, other interluekins, GM-CSF, TNF, INF, and anycombination thereof. The dendritic cells may also stably produce acytokine from a recombinant vector.

Techniques for transferring genes are well known in the art, and any ofthose techniques may be used to produce dendritic cells that stablyproduce the antigen of choice. Preferably, a recombinant retrovirus suchas, for example, an adeno-associated virus (AAV) that has beengenetically manipulated to comprise a portion of an HPV antigen-encodinggene is used to infect monocyte precursors which are then induced todifferentiate into DCs. In a preferred embodiment, an AAV-HPV is used toinfect monocytes which are then induced to differentiate into DCs.

Materials and techniques for the design, generation and production ofrecombinant retroviral vectors and genomes are well known in the art.All such standard DNA materials, techniques and methodologies aresuitable for use herein. An example of a recombinant retroviral vectorsuitable for use herein may comprise the general purpose p5transcriptional promoter to express the antigen genes. Recombinantadeno-associated virus (rAAVs) may be produced by first generating highproducer cell lines to make high-titer virus stocks, as known in theart. As also known by one of skill in the art, these stocks may then beCsCl purified and titered by quantitating the amount of encapsidatedgenomes (virus) by Southern blot.

Suitable non-limiting examples of HPV's useful in the present inventioninclude, HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV58, and anycombination thereof. Preferred HPV's include HPV 16 and HPV 18.Non-limiting examples of combinations include: HPV 16 with at least oneof HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58; HPV 18 with at leastone of HPV16, HPV31, HPV33, HPV35, HPV45, and HPV58; both HPV16 andHPV18 with at least one of HPV31, HPV33, HPV35, HPV45, and HPV58, and atleast any two of HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58.For example, for HPV16 the DCs stably produce HPV 16 E6, HPV 16 E7 orboth. Any portion of the HPV, full-length or not, may be utilizedherein.

The treatment methods of the present invention may be performed on anyorganism having a cancer. Preferably, the methods of the presentinvention are performed on a human. The patient may be positive for CINIII or negative for CIN III. In a preferred embodiment, the screeningmethods are carried out on patients who have tested negative for CINIII, and/or are not afflicted with cervical intraepithelial neoplasia(CIN III). The treatment methods described herein may be useful intreating numerous types of cancer, such as, for example, breast, dermal,oral, penile, vulvar cancer, and any combination thereof. In addition,the treatment methods of the present invention may be against a cancerin any stage of development.

The compositions and methods of the present invention are suitable forany individual afflicted with a cancer. Suitable individuals includemammals such as, humans, dogs, cats, horses, cows, sheep, goats, pigs,rats and mice. As mentioned, preferably the patient is human. Thecompositions and methods of the present invention are also suitable foruse in any tissue or cell line that serves as a model for the study ofcancer. Thus the present invention is useful to medical and health careprofessionals including, medical doctors, and veterinarians, as well asresearch scientists.

It should be noted that the present invention encompasses any and allmethods for screening a patient for a cancer, wherein the methodcomprises detection of an HPV. Any and all methods for treating apatient having a cancer, wherein the method comprises inhibition of anHPV are also within the scope of the invention.

The compositions useful in the methods of the present invention furthercomprise a pharmaceutically acceptable carrier/vehicle. Pharmaceuticallyacceptable carriers/vehicles are known in the art and include aqueoussolutions, non-toxic excipients, including salts, preservatives, buffersand the like, propylene glycol, polyethylene glycol, vegetable oil,injectable organic esters such as ethyloleate, water, saline solutions,parenteral vehicles such as sodium chloride and Ringer's dextrose,glycerol, lipids, alcohols.

Compositions of the present invention may be in any form known in theart, such as an orally digestible form, a sterile injectable form, formssuitable for delayed release, and forms that are enterically coated.Compositions of the invention may be in solid forms, including, forexample, powders, tablets, pills, granules, capsules, sachets andsuppositories, or may be in liquid forms including solutions,suspensions, gels and emulsions.

The compositions and methods of the present invention may beadministered to a recipient/patient as a single dose unit, or may beadministered in several dose units, for a period ranging from one day toseveral years. The dose schedule is dependent upon at least the severityof the glomerular disorder, as well as the mode of administration. Theeffective dose of the compositions of the present invention is furtherdependent upon the body weight (BW) of the recipient/patient and alsoupon the chosen inhibitor. Generally the compositions of the presentinvention are administered orally or intravenously.

Even still another embodiment of the present invention provides for akit for screening a patient for a cancer. Generally the kit comprises aprobe that is specific for the detection of an HPV family member. TheHPV-specific probe may be a single-stranded oligonucleotide sequence, adouble-stranded oligonucleotide sequence, a polypeptide, or anycombination thereof. The HPV may be any HPV family member, non-limitingexamples of which include, HPV16, HPV18, HPV31, HPV33, HPV35, HPV45,HPV58, and any combination thereof. Preferred HPV's include HPV 16 andHPV 18. Non-limiting examples of combinations include: HPV 16 with atleast one of HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58; HPV 18 withat least one of HPV16, HPV31, HPV33, HPV35, HPV45, and HPV58; both HPV16and HPV18 with at least one of HPV31, HPV33, HPV35, HPV45, and HPV58,and at least any two of HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, andHPV58.

The kit of the present invention is useful in screening any organismcapable of developing a cancer. Preferably the sample to be screened isderived from a human patient. The patient may be CIN III positive or CINIII negative, preferably the patient is CIN III negative. The kit of thepresent invention may be useful in detecting a cancer that is in anystage of development, and may be useful in detecting any cancer, suchas, for example breast, dermal, oral, penile, vulvar cancer, and anycombination thereof.

Even yet another embodiment of the present invention provides acomposition for treating a patient having a cancer. Generally, thecomposition comprises an effective amount of an HPV sequence. The sizeof the sequence is not limited. The HPV sequence of the composition maycomprise single-stranded nucleic acids, double-stranded nucleic acids,polypeptides, and any combination thereof. The HPV sequence may be anyone or any combination of HPV family members. Suitable HPV familymembers include but are not limited to, HPV 16, HPV 18, HPV 31, HPV 33,HPV 35, HPV 45, HPV58, and any combinations thereof. Suitablecombinations include: HPV16 and any one of the group consisting of HPV18, HPV 31, HPV 33, HPV 35, HPV 45, and HPV58; HPV 18 and any one of thegroup consisting of HPV 16, HPV 31, HPV 33, HPV 35, HPV 45, and HPV 58;HPV 16 and HPV 18; HPV 16 and HPV 18 and any one of the group consistingof HPV 31, HPV 33, HPV 35, HPV 45, HPV 58, and any combinations thereof;HPV 16, HPV 18 and HPV 33, and any one of the group consisting of HPV31, HPV 35, HPV 45, HPV 58, and any combinations thereof, and at leastany two of the group consisting of HPV 16, HVP 18, HPV 31, HPV 33, HPV45 and HPV 58.

All references cited herein, including research articles, all U.S. andforeign patents and patent applications, are specifically and entirelyincorporated by reference.

EXAMPLES

The following examples are provided to illustrate the present invention.These examples are not intended to and do not limit the scope of theclaims of the present invention, and should not be so interpreted.

Example 1 Analysis of Breast Cancer Tissue for Presence of HPV Via PCR

In the present example, total DNA was isolated from breast cancertissues and analyzed for the presence of HPV by use of PCRamplification. The amplification targeted the L1 gene and was broadspectrum, thus allowing for amplification of many different HPV types.

Patients, Breast Cancer Specimens, and DNA Isolation

17 women with breast cancer receiving examinations and treatment at theUniversity of Arkansas for Medical Sciences (UAMS) from May 1999 toOctober 1999. Portions of needle biopsy tissue was fixed in a PhosphateBuffered Saline: Ethanol (1:1) solution soon after they were acquired.All of the specimens were stored at −80° C. The specimens were processedby grinding, and total cellular DNA was isolated from the specimens bypelleting and resuspending them in lysis buffer (0.5 mg/ml Proteinase K,0.5% SDS, 0.5 mM EDTA, 0.5 mM Tris-HCL, pH7.4). After incubationovernight at 37° C., the total cellular DNA was extracted byphenol/chloroform twice, and then precipitated by ethanol. The DNA wasstored at −80° C. before use. The research has been approved by the UAMSHuman Research Advisory Committee and undertaken in a P2 laboratory.

Polymerase Chain Reaction (PCR) Primer Sets

An HPV L1-targeting consensus primer set has been described elsewhereand was designed to amplify a 450-base segment of HPV L1 gene sequence(Bauer, H. M., et al., JAMA 1991; 265: 472-7). These primers enable PCRamplification of most genital HPV types. A second PCR primer settargeting E6-E7 region has also been described by elsewhere (Fujinaga,Y, et al., J. Gen. Virol. 1991; 72: 1039-1044). These primers allowedfor the amplification of most of the cancer associated HPVs. PCRamplification was carried out as described by Hermonat et al. (HermonatP; et al., Virus Genes 1997; 14:13-17). Positive controls includedvarious amounts of the indicated cloned HPV genome, while negativecontrols included all reagents except specimen DNA.

In order to identify potential contamination from recombinant plasmidscarrying HPV sequences, which are used in the inventors' laboratories,PCR amplification was carried out with pBR322 plasmid targeting primers.The primers were designed to amplify a 414-base segment of pBR322sequence (upstream primer 5′-ATACCTGTCCGCCTTTCTC-3′, and downstreamprimer 5′-AATCTGCTGCTTGCAA AC-3′), containing the origin of replication(ori). The controls included known quantities of pBR322, as low as tenmolecules.

PCR-Dot Blot Hybridization

Amplification of DNA samples was carried out in 100 μl reactions usingapproximately 5 μg of the total cellular DNA, 0.2 mM of each dNTP, 1 μMof each primers, and 2.5 U of Taq iPolymerase according to the suppliersinstructions (Fisher Scientific Co., Pittsburgh, Pa.) instructions.After 5 minutes at 94° C., each sample was subjected to the followingamplification cycle: 55 seconds at 94° C., 1 minutes at 60° C., and 50seconds at 72° C. for 35 cycles, then 10 minutes at 72° C. in the finalcycle.

The dot blotting was carried out with all of the PCR products asdescribed by Hermonat et al. (Hermonat P; et al., Virus Genes 1997;14:13-17). Briefly, 5 μl of PCR products was first denatured by theaddition of 10 μl of 0.4 N NaOH, incubated for 10 minutes, and thenice-bath was done for 5 minutes. After the samples were reneutralized bythe mixing of 200 μl Tris-HCL, pH7.0, 1.5 M NaCL, the samples were addedimmediately to a dot blot apparatus under suction. Multiple nylonmembranes were generated to be analyze by one of several ³²P-labeledprobes. The HPV probes were made with Primer-a-Gene Labeling System(Promega Co.), a random prime labeling kit according to the supplier=sinstructions, which templates were HPV 16, 18 and 31 genomic DNArespectively.

The membranes were analyzed with the radiolabeled full length HPV orpBR322 DNA sequences as indicated. The membranes were soaked inhybridization solution (100 μg of denatured salmon sperm DNA, 1% SDS, 1MNaCL, 10% dextran sulfate) and incubated at 65° C. overnight. Afterhybridization, the membranes were first washed by 2×SSC twice at roomtemperature for 10 minutes, and then washed by 2×SSC and 1% SDS twice at55° C. (super probe) or 65° C. (all others) for 30 minutes.

FIG. 1 provides the results of an assay in which PCR products were dotblotted and probed with an HPV-16/18/31 “super”-probe. As seen in FIG.1, of the breast cancer specimens, six (B2, B4, B10, B13, B15, B17) werepositive for HPV.

Next, the PCR products were probed with a probe specific for HPV-16 only(FIG. 2), a probe specific for HPV-18 only (FIG. 3), and a probespecific for HPV-31 (FIG. 4) only. As can be seen in FIG. 2, the resultsfor the HPV-16-specific probe revealed that specimens B2, B4, and B13gave a strong signal, suggesting that B2, B4, and B13 are most likelyHPV-16. The results from the HPV-18 probe (FIG. 3) revealed that all sixspecimens were positive for HPV-18 (B2, B4, B10, B13, B15, B17), butthat B15, in particular, gave a very strong signal suggesting that thisspecimen was most likely HPV-18. The results for HPV-31 probing (FIG. 4)revealed that all of the six known HPV-positive specimens were weaklypositive. This verifies that the six specimens are HPV positive, butalso further suggests that the specimens are most likely not HPV-31.

The results from a PCR/Dot blot analysis for HPV-16 using an E6-E7junction targeting primer set and probing with HPV-16 sequences areshown in FIG. 5. The dot blotted PCR products were probed for HPV-16 andthe membrane washed at 65° C. Positive controls are at the top. Anunlabeled negative control is one row below on the far left. Note thatthis primer/probe combination appears to pick up the same specimens asidentified by the L12 primer set and HPV-16 probe (compare to FIG. 2)(B2, B4, B13).

The results from a PCR/Dot blot analysis for HPV-18 using an E6-E7junction targeting primer set and probing with HPV-18 sequences areshown in FIG. 6. Positive controls are at the top. An unlabeled negativecontrol is one row below on the far left. Note that this probeidentifies all of the specimens identified by the super probe and the L1products, however B10, and B15 are identified most strongly.

Experiments were then carried out in order to eliminate the possibilityof false positive signal from the tissue sample due to any type ofplasmid contamination. As shown in FIG. 7, these samples were furtheranalyzed for plasmid sequences using PCR primers which targeted theColE1 plasmid origin of replication (ori) region. Essentially allplasmids in use today and in our laboratory are based upon this ori. Adot blot hybridization analysis, probed with ³²P-pBR322 is shown in FIG.7. As can be seen no contamination from plasmid DNA was observed in anyof the samples.

In cervical cancer the HPV DNA is often chromosomally integrated. Todetermine the state of the HPV DNA in breast cancer, 10 μgs of genomicDNA was digested with Bam HI or Xho I. HPV-16 and HPV contain a singleBam HI site, and no Xho I sites. The restricted DNA, along withundigested DNA, were agarose gel electrophoresed, Southern blotted andprobed with ³²P-HPV-16 DNA. The Southern Blot revealed hybridization ofthe probe with an 8 kb band, consistent with episomal DNA. Only in thecervical swab specimen C2 was there significant evidence of chromosomalintegration of the HPV DNA.

Example 2 Construction of the AAV/E6/Neo Genome, Generation of VirusStocks, and Titering of Virus Stocks

The AAV/E6/Neo genome was constructed as a plasmid, in a similar mannerto the construction of the AAV/GM-CSF/Neo viral genome as described byLiu (Liu. Y., et al., J. Inf. Cytok. Res. 2000; 20:21-30), incoporatedherein by reference. However, instead of the GM-CSF gene, the HPV-16 E6open reading frame was cloned by PCR amplification using Pfu polymeraseand ligated into the vector. A structural map of the AAV/E6/Neo vectorused in this study is shown in FIG. 8A. In this construct the E6 gene isexpressed from the AAV p5 promoter, which is known to be active in DC.An AAV/E7/Neo vector was also made in this study (not shown). In the E7construct, the E7 gene is expressed from the AAV p5 promoter.

High titer rough (non-purified) rAAV virus stocks were generated in atwo-step process, using the complementor plasmid ins96-0.8, and titeredas described previously by Hermonat et al., and Li et al. (Hermonat, P.,et al., FEBS Let. 1997: 407:78-84; and Liu. Y., et al., J. Inf. Cytok.Res. 2000; 20:21-30), incoporated herein by reference.) In order togenerate purified rAAV virus, the technique described by Auricchio etal. was used (Human Gene Therapy 2001; 12:71-76). Briefly, the virussolution treated by DNase I (Promega Co.) was incubated with 0.5%deoxycholic acid (Sigma Co.) for 30 minutes at 37° C. After filterationthe solution was applied on a heparin-agarose column (Sigma Co.). Thematrix was washed twice with 25 ml of 0.254M NaCl-PBS, pH 7.4, and theneluated with 15 ml of 0.554M NaCl-PBS, pH 7.4. The eluate was thenconcentrated to about 1 ml using a Millipore Biomax-100K NMWL filterdevice and cetrifugation.⁵⁰. Purity of the viral preparation (100 ul)was assessed on 4-20% SDS-polyacrylamide gel run. The proteins weredetected by Coomassie staining. The titer of purified virus wascalculated by dot blot and determined to be 1×10¹¹ encapsidated genomesper ml.

AAV/E6/Neo virus stock was generated by the two step process mentionedabove, and a comparison of various G418 resistant producer cell lines,by dot blot hybridization, is shown in FIG. 8B. The titering of thenon-purified virus stock, in encapsidated genomes (eg) per ml of thisvirus stock, by dot blot hybridization is shown in FIG. 8C (about 10¹¹eg/ml).

Cells Used in this Study

The primary cervical cancer cell lines, CA1 (patient 1) and CA2 (patient2), have been described previously, both containing HPV-16 DNA, and wereapproximately 10 passages from initial isolation (Santin, A. D., et al.,J. Virol., 1999: 73: 5402-5410). These cells were grown inKeratinocyte-SFM supplemented with epidermal growth factor and bovinepituitary extract (Gibco BRL/Life Technologies). Human Leucocyte Antigen(HLA) typing of these cells gave haplotypes of HLA A1 for both CA1 andCA2, respectively. Mo and DC were derived from peripheral bloodmononuclear cells (PBMC). PBMC were separated by routine Ficoll gradientmethod from fresh blood drawn from healthy persons. The normal donor hada haplotype of HLA A1, compatible with the target cancer cells. ThePBMCs were inoculated into six-well culture plates and incubated withtwo milliliters of AIM-V medium for two hours at 37° C. and 5% CO₂. Atthat time non-adherent cells were removed by carefully washing themonolayer three times with phosphate buffered saline (PBS, pH 7.0).

Infection of Mo/DC with AAV Virus and Treatment with Cytokines.

Immediately after the removal of the non-adherent cells, the adherent Mowere infected (pulsed) with 0.5 ml of virus stock (˜5×10¹⁰ encapsidatedgenomes) when using the non-purified virus, or 10⁷ encapsidated genomeswhen using the purified virus. After two hours incubation themedium/virus solution was removed, the cells were washed with AIM-V, andfinally fed with AIM-V medium. The infection protocol is outlined inFIG. 8D. The Mo/DC precursors were infected with 0.5 ml of virus stockor lysate at days 0, 3, and 5. Throughout this time period the Mo/DCculture was treated with human GM-CSF (LEUKINE®, Immunex Corporation,1.4×10⁶ IU/250 μgs) at a final concentration of 800 IU/ml. At day three,to induce the maturation of Mo into DC, human interleukin 4 (IL-4, R & DSYSTEMS Co.) at 1000 IU/ml was added to the medium. Finally, at day 5,recombinant human interleukin 2 (IL-2, R & D SYSTEMS Co.) at 10 U/ml wasadded.

Generation of Bacterial E6 Protein and Lipofection of Mo/DC.

GST-E6 protein was generated in a similar manner to previous generationof GST-E7.³⁴ The Mo/DC were lipofected (pulsed) with 15 μg of GST-E6 onday 5 as previously described (Santin, A. D., et al., J. Virol., 1999:73: 5402-5410). The treatment of the protein pulsed Mo/DC with cytokineswas the same as the virus infected DC.

mRNA Isolation and RT-PCR Analysis for E6 Expression.

E6 mRNA expression was measured by RT-PCR amplification along with acellular mRNA control. Total RNA was isolated from mock (lysate)infected and AAV/E6/Neo infected Mo/DC using Trizol reagent (GIBCO BRLLife Technologies Inc.), according to the manufacturer's protocol andtreated with 5 U/μg of RNase-free DNase I (Promega Co.) at 37° C. for 1hour. Messenger RNA was then separated using the Oligotex mRNA Mini Kit(QIAGEN Inc.) according to the supplier's instruction. The first-strandcDNA synthesis was performed at 37° C. for 1 hour in a final volume of25 μl reaction buffer [1 μg mRNA; 50 mM Tris-HCl, pH8.3; 75 mM KCl; 3 mMMgCl₂; 10 mM DTT; 0.5 μg oligo(dT)₁₅ (Promega Co.); 0.5 mM each of thefour dNTPs; 30 U of RNasin (Promega Co.) and 200 U of M-MLV ReverseTranscriptase RNase H Minus (Promega Co.)]. PCR amplification of thecDNA was performed in 100 μl reaction volume which contained 2.5 U TaqDNA polymerase (Fisher Scientific Co.); 10 mM Tris-HCl₁, pH8.3; 50 mMKCl; 2 mM MgCl₂; 0.2 mM each of the four dNTPs; 1 μM of each upstreamand downstream primer specific for the cDNA template and 10 μl cDNAtemplate. The E6 primer set used was 5′-ACCACAGTTATGCACAGAGC-3′ and5-AGGACACAGTGGCTTTTGAC-3′, which targeted amplification of the HPV-16sequences from nt 139 to 420. A control RT-PCR analysis of expression ofthe housekeeping gene TFIIB was also undertaken with the primer set5′-GTGAAGATGGCGTCTACCAG-3′ and 5′-GCCTCAATTTATAGCTGTGG-3′, whichamplified nt 356-1314 of that mRNA. To insure that DNA wasn'tcontributing to the results, a direct PCR was also undertaken. Theproducts were then analyzed on an agarose gel, stained with ethidiumbromide, and visualized by ultraviolet light.

In these studies three infections were undertaken as indicated in FIG.8D. After AAV infection and GM-CSF treatment, at day three, the cellswere finally treated with IL-4 to induce differentiation into DC. Thetransduction of the Mo/DC population was analyzed by observing RNAexpression of the E6 transgene. Polyadenylated RNA was isolated fromAAV/E6/Neo infected and mock-infected DC cultures at day 10 (after IL-4introduction and differentiation into DC) and was analyzed for E6 mRNAby RT-PCR. Expression of the cellular TF_(II)B gene was also undertakenas a control. The results, shown in FIG. 9 demonstrate that E6expression only takes place in the virally infected DC. In FIG. 9, thepositive contro was the PCR product resulting from the Aav/E6/NEO vectorplasmid as a template. Another control was PCR analysis of RNA fromcells infected by AAV/E6/Neo virus. A final control included theanalysis of cellular TF_(II)B RNA. Note that only RNA from cellsinfected with AAV/E6/Neo virus resulted in an appropriate E6 RT-PCRsized product, while mock and PCR amplification of the RNA from cellsinfected by AAV/E6/Neo virus did not give a product, indicating lack ofcontaminating DNA.

Intracellular Staining for E6.

The protocol used was adapted from that described by Pala et al.,(Immunology 2000; 100:209-216). The Mo/DC were infected with virus orlipofected with protein as described above. Cells were then treated withIL-4 and GM-CSF under standard conditions. Seven days afterinfection/lipofection the cells were harvested, washed and fixed with 2%paraformaldehyde in PBS for 20 min at room temperature. The cells werewashed and permeabilized with PBS/1% BSA/0.5% saponin (S-7900, Sigma)for 10 min at room temperature. Cells were stained with anti-HPV-16/18E6 (Chemicon Inc., Temecula, Calif.; Cat no MAB874) plusFITC-anti-mouse-Ig (Becton Dickinson Inc., cat no 554001) and analyzedby flow cytometry.

The efficiency of virus/gene and lipofection/protein-pulsing of Mo/DCwas analyzed and compared by intracellular staining. The cells wereanalyzed four days after pulsing. The results, shown in FIG. 10,demonstrate that AAV/E6/Neo infection of Mo resulted in a much higherpercentage of cells containing intracellular E6 protein than directprotein-pulsing (72% to 29%). FIG. 10A provides the results fromlipofection, 10B provides the results from infection. Mo were pulsed(infection or lipofection) as indicated, treated with cytokines, andanalyzed for E6 protein by intracellular staining on day 4 as describedin the Materials and Methods section. Note that AAV/E6/Neoinfection/pulsing gave the highest levels of E6 positive cells comparedto protein lipofection (72% versus 29%)

Detection of Viral Integration by PCR/Southern Blot Analysis.

Chromosomal integration of the AAV/E6/Neo genome was undertaken byvector-chromosome junction PCR amplification and Southern blot analysisas previously described (Liu. Y., et al., J. Inf. Cytok. Res. 2000;20:21-30). Chromosomal integration of the AAV/E6/Neo vector in DC wasobserved. Chromosomal integration, while not essential for geneexpression from AAV vectors, does signifies a permanent geneticalteration of the DC, and is a desirable “gold standard” for viraltransduction. Chromosomal integration was demonstrated by PCRamplification of vector-chromosome junctions using primers complementaryto the SV40 promoter within the vector and Alu I repetitive chromosomalelements. DC were similarly treated as in the RNA analysis experimentsof FIG. 9. Junction products were analyzed by agarose gelelectrophoresis, Southern blotted, and probed for Neo sequences.Briefly, total cellular DNA from the infected, CD83+ selected cells anduninfected cells served as template in PCR amplification assays usingprimers targeting the SV40 early promoter of the vector and the cellularrepetitive Alu I element. The products were Southern blotted and probedwith ³²P-Neo DNA, shown in FIG. 11. The positive control lane contained100 ng of Eco RV digested AAV/GM-CSF/Neo plasmid (6.7 & 1.3 Kb). Thenegative control lane contained products from a PCR reaction with DNAmock infected cells. Note that multiple Neo-positive bands result fromthe infected DC population indicating chromosomal integration by thevector, and that multiple vector-positive cell clones are present in thepopulation. As shown in FIG. 11, multiple vector-chromosomal junctionproducts were observed in the AAV/E6/Neo infected DC, but not in mockinfected DC. Unfortunately, the vector must integrate close to an Alu Ielement in order to be identified by this technique. In any case, thesedata indicate that at least some of the viral genomes are able tochromosomally integrate in the DC population.

Chromium Release Assay of CTL Activity.

Non-adherent PBMCs (T cells and B cells) were washed and resuspended inAIM-V at 10-20×10⁶ cells/well in 6-well culture plates (Costar,Cambridge, Mass.) with rAAV or GST-E6 pulsed DC (ratios from 20:1,responders:dendritic). The cultures were supplemented with recombinanthuman GM-CSF (500 U/ml) and recombinant human IL-2 (10 U/ml). At 7 dayspost-addition of the PBMC, the cells were assayed for tumor cell killingactivity in a 6-hour chromium-51 release assay as previously described(Santin, A. D., et al., J. Virol., 1999: 73: 5402-5410). One of two HLAclass I A1 compatible primary cervical tumor cells was ⁵¹Cr-labeled andused as targets as previously described (Santin, A. D., et al., J.Virol., 1999: 73: 5402-5410). To determine the structures on the targetcells involved in lysis, monoclonal anti-Class I Mabs were used to blockcytotoxicity. The ⁵¹Cr-labeled tumor targets were pre-incubated withMabs specific for monomorphic HLA class I W6/32 (50 ug/ml) (hybridomaobtained from the ATCC, Rockville, Md.). The effector cells and⁵¹Cr-labeled targets were then incubated in a final volume of 200 ul for6 hours at 37° C. with 5% CO₂.

Cell Surface Marker Analysis of T Cells and DC by Flourescent AntibodyCell Sorting (FACS).

For the analysis of T cells, at day 12 of the experiment the primed Tcell populations were analyzed for surface markers. A panel of mAbsrecognizing the following antigens was used: anti-CD4, anti-CD8,anti-CD56 (Pharmingen, San Diego, Calif.). Control irrelevantisotype-matched FITC- or PE-conjugated mAbs were obtained fromBecton-Dickinson. These cells were greater than 95% viable as assessedby trypan blue exclusion. Cell suspensions were counted and distributedinto 12×75 mm tubes. Mouse monoclonal antibodies were diluted in coldassay buffer and the final pellet was resuspended in 500-μl volume.Tubes were incubated for 30 minutes followed by two washes with assaybuffer and the final cell pellet was resuspended in 500 μls of assaybuffer for subsequent analysis. Cells were analyzed with a fluorescenceactivated cell sorter (FACS; Becton-Dickinson) with a 15 mW argon laserwith an excitation of 488 nm. Fluorescent signals were gated on thebasis of cell dimension (i.e. forward and right angle light scatteringtypical of PBL activated. Gated signals (5,000-10,000) were detected at585 BP filter and analyzed using Cell Quest software (Becton-Dickinson).

For the analysis of DC, a panel of mAbs recognizing the followingantigens was used: anti-CD40 (Immunotech, Marseille, France); anti-CD14,anti-DR, anti-CD80 (Becton-Dickinson), anti-CD86 (Pharmingen, San Diego,Calif., USA), anti-CD83 (Coulter, Miami, Fla., USA). Control irrelevantisotype-matched FITC- or PE-conjugated mAb were obtained fromBecton-Dickinson. Briefly, nonadherent cells were harvested by washingthe plates with phosphate buffered saline (PBS pH 7.2, Gibco). Adherentcells were recovered by incubating the plates at room temperature for15-20 minutes in the presence of Cal+ and Mg′+-free PBS, followed bygentle scraping. These cells were >95% viable as assessed by trypan blueexclusion. Cell suspensions were counted and distributed into 12×75 mmtubes. Mouse monoclonal antibodies were diluted in cold assay buffer(PBS, pH 7.2, supplemented with 0.I % FBS) and added in a 50 μl volume.For direct fluorescence, tubes were incubated for 30 min followed by twowashes with assay buffer and the final cell pellet was resuspended in500 μl assay buffer for subsequent analysis.

AAV-Mediated Pulsing of DC Results in Rapid and Effective T CellPriming.

With strong evidence of DC transduction and expression by the AAV/E6/Neovector, the ability of the AAV/E6/Neo vector to antigenically pulse DCswas analyzed. Adherent Mo were mock, GST-E6, or AAV/E6/Neo virus pulsedas before (FIG. 8D). Each of these cultures were then treated withGM-CSF and IL-4 as prescribed by Sallusto and Lanzavecchia (1994) andRomani et al. (1994) for generating DC. At day 5 the resulting DCs werethen incubated with non-adherent peripheral blood lymphocytes. At daytwo of IL-4 treatment, representative pictures were taken of thecultures at low and high power. Note that the virus treated DC-T cellcultures exhibited much higher levels of rosetted cell clusters,suggesting stronger DC-T cell interaction.

As AAV/antigen pulsing is novel, the cultures were observed on a dailybasis. It was almost immediately noticed that cell clustering was takingplace in an enhanced manner in the AAV-pulsed DC cultures relative tothe protein-pulsed or lysate control cultures (FIG. 12). Upon examiningthe cell morphology present, the present inventors believe theseclusters to be due to T cell-DC resetting. This led to the speculationby the inventors that the AAV-pulsing of DC might be allowing for morerapid priming of the T cells. After 7 days of priming the rosettes weredispersing. This change was interpreted as signaling the completion ofpriming and the subsequent death of the DC as a target. This is in sharpcontrast to the protein- and lysate pulsed cultures which still did notshow extensive resetting. Normally DC-T cell incubation and primingrequire 2 to 3 weeks to allow for significant cytotoxic T lymphocyteactivity (CTL). AAV-pulsing of DCs may require only 7 days of priming.

The ability of these 7 day-primed T cells to carry out class Irestricted recognition and killing of HLA A1 matched primary cervicalcancer cells was tested in chromium release assays. The previouslycharacterized primary cervical cancer cell line 1, CA-1, was used as atarget. Our impressions of rapid priming was fully borne out by thelevel of target killing. A representative experiment is shown in FIG.13. FIG. 13A Shows a representative experiment of cytotoxic responseresulting from the indicated pulsing techniques Mo/DC, and T cells froma normal individual against HLA A1 matched primary cervical cancer cells(CA1) and a primary multiple myeloma (MM). Note that the addition of theclass I blocking antibody W632 greatly inhibits killing. Also note lackof killing activity against the primary MM. FIG. 13B shows arepresentative experiment of cytotoxic response against a second HLA A1matched primary cervical cancer (CA2). Again, note that the addition ofthe class I blocking antibody W632 greatly inhibits killing.

Normally, protein/DOTAP pulsing of DC results in significant targetkilling. However, by allowing only a short period of priming, thepercent killing resulting from this technique was only slightly abovethe cell lysate control (mock). In sharp contrast, theAAV/E6/Neo-pulsed-DC-primed T cells resulted in much higher killingactivity than the protein primed and lysate controls. These sameAAV/E6/Neo primed cells were unable to lysis an unrelated multiplemyeloma target. Finally, the addition of the anti-class I antibody W632significantly blocked killing, indicating class I restriction ofkilling. We next assayed for class I restricted killing of a second HLAA1 matched, previously characterized primary cervical cancer cell lineCA-2 (Santin, A. D., et al., J. Virol. 1999; 73: 5402-5410). Theresulting killing was very similar to that against CA-1 (FIG. 13).Again, the addition of the anti-class I antibody W632 significantlyblocked killing, indicating class I restriction of killing. Finally,because non-purified virus was utilized in these experiments whichcontained lysed cellular components, the CTL experiment was repeatedusing a heparin column-purified virus stock. DC were pulsed with 10⁷purified encapsidated genomes of AAV/E6/Neo (approximately 10⁻³ virusused in the FIG. 13 experiments). A CTL assay was then carried out onthe CA1 cervical cancer primary target as described in FIG. 13. Theresulting killing for AAV/E6/Neo, AAV/E6/Neo plus anti-class Iantibodies, GST-E6, and lysate control pulsing of DC was 36.1+/−1.4%,0.4+/−0.3%, 3.1+/−1.4%, and 2.0+/−0.5%, respectively. These dataindicate that the CTL activity in all of these experiments was in factdue to AAV/E6/Neo viral transduction/pulsing of DC.

Example 3 Higher CD8/CD4 and Lower CD56/CD8 Cell Ratios Result withAAV-Mediated Pulsing/Priming

The makeup of the T cell populations, which resulted fromAAV-transduction or protein lipofection, was observed. An effective CTLresponse, while requiring CD8+ T cells as an effector of lysis, alsorequires CD4+ helper T cells. Flow cytometric analysis was used todetermine the phenotype of the population of the lysate, GST-E6 pulsed,and AAV/E6/Neo pulsed T cell populations.

FIG. 14A shows the CD8 and CD4 prevalence within the primed populationresulting from three different techniques as indicated (on the right),as well as an FL1-H, FL2-H control (left). FIG. 14B shows the CD56 andCD8 ratios in the same experimental situations as A.

As shown in FIG. 14A, in the mock case, one sees a normal ratio of CD8to CD4 postive cells (1.21:1). In the GST-E6 pulsing case, the D8/CD4ratio remains the same (1.23:1). In sharp contrast in the AAV/E6/Neopulsed case, the ratio of CD8/CD4 changes dramatically to 7.0:1.Normally, one would not expect this high ratio to arise until threeweeks of DC/T cell priming suggesting that the AAV-Ag pulsing of DCresults in not only quicker activation, but also higher killingactivity, HLA class I restricted, on a per CD8+ T cell basis. The highkilling is consistent with a Type 1 (Th 1) response. This is importantbecause recent studies have suggested that progression to cervicalcancer from precursor lesions may be associated with a preferential Type2 (Th 2) T cell response, along with significant dysfunction of Type 1 Tcell response in patients with high grade cervical intraepitheliallesions and invasive cervical cancer (Cleric M., et al., J. Natl. Can.Inst. 1997; 90: 261-3).

The expression of CD56 was also observed. Some consider this marker asbeing specific for natural killer T cells. However, others have reportedthat some CD8+ T cells do express CD56 and do exhibit HLA class Irestricted killing. In spite of this confusion, all of T cellpopulations tested herein exhibited low CD56 expression. However, whatis noteworthy in this analysis is the very high level of CD8+ T cells inthe case of AAV/E6/Neo pulsing, confirming the CD8/CD4 analysis (FIG.14A). Taken together, these data indicate a very different resultingprimed T cell population when AAV-antigen pulsing was used, and suggeststhat the increased killing activity may be a result of these changes inthe T cell population These data also indicate that CD8 is a specificlineage marker in HLA class I restriction in our system.

Example 4 AAV/E6 Vector-Pulsing Results in DC with Higher CD80, andLower CD86 Expression

Finally, the DC resulting from the various pulsing techniques wascharacterized to observe if significant differences were discernable.Flow cytometric analysis was used to determine the phenotype ofuntreated, lysate-pulsed, GST-E6-pulsed, and AAV/E6/Neo-pulsed DCpopulations. The results, shown in FIGS. 15 and 16, demonstrate that theDC generated from all for techniques share all of the common DC markers.However, AAV/E6/Neo pulsed DC did express significantly higher levels ofCD80 and lower levels of CD86 than the GST-E6-pulsed DC. In FIG. 15, Mowere treated as indicated, treated with GM-CSF and IL-4 and analyzed byFACS for mean flourescent intensity (MFI) on day 7. “−” no detectableMFI staining, “+”=MFI 10¹-10². “++”=MFI 10²-10³. “+++”=MFI 10³-10⁴.Representative of three experiments. Note AAV-pulsing results in higherCD80 and lower CD86 levels compared to protein pulsing. FIG. 16 provideshistograms of CD80 expression complied in FIG. 13. FIG. 16A provides thesize analysis of general DC populations, and FIG. 16B provides the DCCD80 expression under different conditions.

While the illustrative embodiments of the invention have been describedwith particularity, it will be understood that various othermodifications will be apparent to and can be readily made by thoseskilled in the art without departing from the spirit and scope of theinvention. Accordingly, it is not intended that the scope of the claimsappended hereto be limited to the examples and descriptions set forthherein but rather that the claims be construed as encompassing all thefeatures of patentable novelty which reside in the present invention,including all features which would be treated as equivalents thereof bythose skilled in the art to which this invention pertains.

1. A method of screening a patient for cancer, the method comprising: a)assaying for the presence of human papillomavirus in a sample from abiopsy taken from a patient, wherein the patient has been determined tobe negative for CIN III, wherein said assaying comprises amplifyinghuman papillomavirus gene sequences by use of a pair of primers toproduce an amplified product, and probing said amplified product for thepresence of a portion of an HPV sequence, wherein said pair of primersis either SEQ ID NO: 1 and SEQ ID NO: 2, or SEQ ID NO: 3 and SEQ ID NO:4, and wherein presence of said HPV is indicative of breast cancer insaid patient.
 2. The method of claim 1 wherein the biopsy is obtained byperforming the technique of ductal lavage on a breast of a patient. 3.The method of claim 1 wherein the patient is a human, wherein the canceris in any stage of development, and wherein the cancer is selected fromthe group consisting of breast, dermal, oral, penile, vulvar cancer, andany combination thereof.
 4. The method of claim 1 wherein saidamplifying is achieved by use of polymerase chain reactionamplification.
 5. The method of claim 1 wherein said amplifying isachieved by use of reverse-transcription polymerase chain reactionamplification.
 6. The method of claim 1 wherein said HPV sequence isselected from the group consisting of HPV18, HPV31, HPV 33, HPV35,HPV45, HPV58.
 7. The method of claim 1 wherein said probing is for thepresence of at least two HPV sequences selected from the groupconsisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
 8. The methodof claim 7 wherein one of the at least two HPV sequences is HPV18. 9.The method of claim 1 wherein said probing is for the presence of aportion of HPV16 and at least one HPV sequence selected from the groupconsisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
 10. A method ofscreening a patient for cancer, the method comprising: a) assaying forthe presence of human papillomavirus in a sample from a biopsy takenfrom a patient wherein said assaying comprises amplifying humanpapillomavirus gene sequences by use of a pair of primers to produce anamplified product, and probing said amplified product for the presenceof a portion of an HPV sequence selected from the group consisting ofHPV18, HPV31, HPV 33, HPV35, HPV45, HPV58, wherein said pair of primersis either SEQ ID NO: 1 and SEQ ID NO: 2, or SEQ ID NO: 3 and SEQ ID NO:4, and wherein presence of said HPV is indicative of breast cancer insaid patient.
 11. The method of claim 9 wherein said probing is for thepresence of at least two HPV sequences selected from the groupconsisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
 12. The methodof claim 9 wherein the patient is a human, wherein the cancer is in anystage of development, and wherein the cancer is selected from the groupconsisting of breast, dermal, oral, penile, vulvar cancer, and anycombination thereof.
 13. The method of claim 9 wherein the biopsy isobtained by performing the technique of ductal lavage on a breast of apatient.
 14. The method of claim 9 wherein said amplifying is achievedby use of polymerase chain reaction amplification.
 15. The method ofclaim 9 wherein said amplifying is achieved by use ofreverse-transcription polymerase chain reaction amplification. 16-20.(canceled)
 21. A method of screening a patient for a cancer, the methodcomprising: a) contacting cellular material with an HPV specific probe,wherein the cellular material is extracted from a biopsy taken from apatient, and wherein the patient has been determined to test negativefor CIN III.
 22. The method of claim 21 wherein the cellular material isderived from cells obtained by performing the technique of ductal lavageon a breast of a patient.
 23. The method of claim 21 wherein thecellular material comprises nucleic acid, polypepetides, or acombination thereof.
 24. The method of claim 21 wherein the probe is anHPV DNA or RNA oligonucleotide sequence complementary to the plus strandof an HPV DNA sequence.
 25. The method of claim 21 wherein the probe isan HPV DNA or RNA oligonucleotide sequence complementary to a portion ofan HPV mRNA sequence.
 26. The method of claim 21 wherein the probe is anHPV DNA or RNA oligonucleotide sequence complementary to a portion of anHPV ribosomal RNA sequence.
 27. The method of claim 21 wherein the probeis an antibody specific to an epitope of an HPV protein.
 28. The methodof claim 27 wherein the protein is HPV16 E6 or HPV16 E7.
 29. The methodof claim 21 wherein the HPV is selected from the group consisting ofHPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
 30. The method of claim 21wherein step a) further comprises contacting the cellular material witha second HPV specific probe, wherein the first and second HPV aredifferent from one another and are selected from the group consisting ofHPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
 31. The method of claim 21wherein step a) further comprises contacting the cellular material witha second HPV specific probe, wherein the first HPV specific probe isspecific to HPV 16 and the second HPV specific probe is specific to atleast one HPV selected from the group consisting of HPV18, HPV31, HPV33, HPV35, HPV45, HPV58.
 32. The method of claim 21 wherein the canceris in any stage of development, and wherein the cancer is selected fromthe group consisting of breast, dermal, oral, penile, vulvar cancer, andany combination thereof.
 33. A method of screening a patient for acancer, the method comprising: a) contacting cellular material with aprobe specific to a first HPV, and a second probe specific to a secondHPV, wherein the cellular material is extracted from a biopsy taken froma patient and wherein the first HPV is HPV 16, and the second HPV isselected from the group consisting of HPV18, HPV31, HPV 33, HPV35,HPV45, HPV58.
 34. The method of claim 33 wherein the cellular materialis derived from cells obtained by performing the technique of ductallavage on a breast of a patient.
 35. The method of claim 33 wherein thecellular material comprises nucleic acid, polypepetides, or acombination thereof.
 36. The method of claim 33 wherein the probe is anHPV DNA or RNA oligonucleotide sequence complementary to the plus strandof an HPV DNA sequence.
 37. The method of claim 33 wherein the probe isan HPV DNA or RNA oligonucleotide sequence complementary to a portion ofan HPV mRNA sequence.
 38. The method of claim 33 wherein the probe is anHPV DNA or RNA oligonucleotide sequence complementary to a portion of anHPV ribosomal RNA sequence.
 39. The method of claim 33 wherein the probeis an antibody specific to an epitope of an HPV protein.
 40. The methodof claim 36 wherein the protein is HPV16 E6 or HPV16 E7.
 41. The methodof claim 33 wherein the cancer is in any stage of development, andwherein the cancer is selected from the group consisting of breast,dermal, oral, penile, vulvar cancer, and any combination thereof.
 42. Amethod of screening a patient for a cancer, the method comprising: a)contacting cellular material with a probe specific to a HPV selectedfrom the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58,wherein the cellular material is extracted from a biopsy taken from apatient.
 43. The method of claim 37 wherein step a) further comprises asecond probe specific to a second HPV selected from the group consistingof HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58, wherein the first andsecond HPV are different from one another.
 44. The method of claim 42wherein the cellular material is derived from cells obtained byperforming the technique of ductal lavage on a patient.
 45. The methodof claim 42 wherein the cellular material comprises nucleic acid,polypepetides, or a combination thereof.
 46. The method of claim 42wherein the probe is an HPV DNA or RNA oligonucleotide sequencecomplementary to the plus strand of an HPV DNA sequence.
 47. The methodof claim 42 wherein the probe is an HPV DNA or RNA oligonucleotidesequence complementary to a portion of an HPV mRNA sequence.
 48. Themethod of claim 42 wherein the probe is an HPV DNA or RNAoligonucleotide sequence complementary to a portion of an HPV ribosomalRNA sequence.
 49. The method of claim 42 wherein the probe is anantibody specific to an epitope of an HPV protein.
 50. The method ofclaim 42 wherein the cancer is in any stage of development, and whereinthe cancer is selected from the group consisting of breast, dermal,oral, penile, vulvar cancer, and any combination thereof.
 51. A methodof treating a patient comprising: a) administering a compositioncomprising an effective amount of an antisense HPV sequence to apatient.
 52. The method of claim 51 wherein administering comprisesdelivery of the composition into a milk duct of a breast of the patientby insertion of a microcatheter into a nipple surface orifice of saidbreast.
 53. The method of claim 51 wherein the HPV is selected from thegroup consisting of HPV16, HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58,and any combination thereof.
 54. The method of claim 51 wherein theantisense HPV sequence is expressed from a viral expression vector. 55.The method of claim 51 wherein the patient is human and has a cancer inany stage of development.
 56. The method of claim 51 wherein the canceris breast, dermal, oral, penile, or vulvar cancer, or any combinationthereof.
 57. A method of treating a patient comprising: a) administeringan effective amount of a composition to a patient, wherein thecomposition comprises an agent that inhibits expression of at least oneHPV gene.
 58. The method of claim 57 wherein administering comprisesdelivery of the composition into a milk duct of a breast of the patientby insertion of a microcatheter into a nipple surface orifice of saidbreast.
 59. The method of claim 57 wherein the agent is anoligonucleotide comprising antisense HPV DNA, RNA or ribosomal RNA. 60.The method of claim 57 wherein the agent is an oligonucleotidecomprising sequences complementary to the plus or minus strand of HPVDNA.
 61. The method of claim 57 wherein the HPV is selected from thegroup consisting of HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV58, andany combination thereof.
 62. The method of claim 57 wherein the patientis human and has a cancer in any stage of development.
 63. The method ofclaim 57 wherein the cancer is breast, dermal, oral, penile, or vulvarcancer, or any combination thereof.
 64. A method of treating a patientcomprising: a) administering an effective amount of a compositioncomprising an agent that specifically inhibits the HPV16 E6 protein orthe HPV16 E7 protein.
 65. The method of claim 64 wherein administeringcomprises delivery of the composition into a milk duct of a breast ofthe patient by insertion of a microcatheter into a nipple surfaceorifice of said breast.
 66. The method of claim 64 wherein the agent isan antibody specific for the HPV16 E6 protein or HPV16 E7 protein. 67.The method of claim 64 wherein the patient is human and has a cancer inany stage of development.
 68. The method of claim 64 wherein the canceris breast, dermal, oral, penile, or vulvar cancer, or any combinationthereof.
 69. A method of treating a patient comprising: a) transfectingdendritic precursor cells of a patient with a recombinant viral vectorthat drives expression of an HPV antigen; b) treating the dendriticprecursor cells with a cytokine to produce dendritic cells stablyexpressing the HPV antigen; c) contacting T cells together with thedendritic cells stably expressing the HPV antigen to produce primed Tcells; and d) administering to the patient an effective amount of eitherthe primed T cells, dendritic cells, or a combination thereof.
 70. Themethod of claim 69 wherein the cytokine is selected from the groupconsisting of interluekins, GM-CSF, TNF, and any combination thereof.71. The method of claim 69 wherein the patient is human, and wherein thepatient has a cancer in any stage of development.
 72. The method ofclaim 71 wherein the cancer is breast, dermal, oral, penile, or vulvarcancer, or any combination thereof.
 73. The method of claim 69 whereinthe recombinant viral vector is an adeno-associated viral vector. 74.The method of claim 69 wherein the HPV is selected from the groupconsisting of HPV16, HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58, and anycombination thereof.
 75. The method of claim 69 wherein the HPV antigenis HPV E6 or HPV E7.
 76. A kit for screening a patient for a cancer, thekit comprising: a) a probe specific for detection of an HPV.
 77. The kitof claim 77 wherein the probe is a single-stranded olidonucleotidesequence, a double-stranded oligonucletide sequence, a polypeptide, orany combination thereof.
 78. The kit of claim 77 wherein the HPV isselected from the group consisting of HPV16, HPV18, HPV31, HPV35, HPV45,HPV58, and any combination thereof.
 79. The kit of claim 77 wherein thepatient is human, wherein the cancer is in any stage of development, andwherein the cancer is selected from the group consisting of breast,dermal, oral, penile, vulvar cancer, and any combination thereof.
 80. Acomposition for treating a patient having a cancer, the compositioncomprising: an effective amount of an HPV sequence.
 81. The compositionof claim 80 wherein the sequence is selected from the group consistingof single-stranded nucleic acids, double-stranded nucleic acids,polypeptides, and any combination thereof.
 82. The composition of claim80 wherein the HPV sequence is selected from the group consisting of HPV16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 45, HPV58, and any combinationsthereof.
 83. The composition of claim 80 wherein the HPV sequence isHPV16 and any one of the group consisting of HPV 18, HPV 31, HPV 33, HPV35, HPV 45, HPV58, and any combinations thereof.
 84. The composition ofclaim 80 wherein the HPV sequence is HPV 18 and any one of the groupconsisting of HPV 16, HPV 31, HPV 33, HPV 35, HPV 45, HPV58, and anycombinations thereof.
 85. The composition of claim 80 wherein the HPVsequence is a combination of HPV 16 and HPV
 18. 86. The composition ofclaim 80 wherein the HPV sequence is a combination of HPV 16 and HPV 18and at least any one of the group consisting of HPV 31, HPV 33, HPV 35,HPV 45, HPV58, and any combinations thereof.
 87. The composition ofclaim 80 wherein the HPV sequence is a combination of HPV 16, HPV 18 andHPV 33, and at least any one of the group consisting of HPV 31, HPV 35,HPV 45, HPV58, and any combinations thereof.
 88. A method of screening apatient for breast cancer, the method comprising: a) assaying for thepresence of human papillomavirus in a sample from a biopsy taken from apatient, wherein said assaying comprises amplifying human papillomavirusgene sequences by use of a pair of primers to produce an amplifiedproduct, and probing said amplified product for the presence of HPV 16and a second human papillomavirus selected from the group consisting ofHPV18, HPV31, HPV 33, HPV35, HPV45, HPV58, wherein said pair of primersis SEQ ID NO: 3 and SEQ ID NO: 4, and wherein presence of HPV 16 andsaid second human papillomavirus is indicative of breast cancer in saidpatient.
 89. The method of claim 88 wherein the patient is a human, andwherein the cancer is in any stage of development.
 90. The method ofclaim 88 wherein the biopsy is obtained by performing the technique ofductal lavage on a breast of a patient.
 91. The method of claim 88wherein said amplifying is achieved by use of polymerase chain reactionamplification.
 92. The method of claim 88 wherein said amplifying isachieved by use of reverse-transcription polymerase chain reactionamplification.